I'm seeking advice for how to treat tissue samples to ensure that most of the proteins are solubilized, to improve the consistency of my protein quantitation results.
I am using the Bradford assay to quantitate my protein samples, which I use to normalize lipid data. I am seeing large variability in results, and by comparing the mass of the tissue with the protein concentrations, I think my problem is that I am not solubilizing all of the protein in my sample. I suspect the problems could be inconsistent homogenization, a non-optimal centrifugation step, or using an insufficient buffer.
I am working with planarian (flatworm) tissue. Based on C. elegans papers, I add 2 large worms (about 50 mg of tissue) and 1 mL PBS into a centrifuge tube, and use a pestle to dissociate the tissue. Then I pellet the tissue at 16000g for 5 minutes - this produces a sizable pellet (it looks like this contains the body pigments, the tough pharyngeal tissue, and cell debris). I reserve the supernatant and use it as-is for the Bradford assay.
I'm afraid that a lot of the protein may be going into the pellet! Can anyone advise how I can optimize this protocol to ensure thorough protein solubilization? E.g. add sonication, or spin at lower g's, or use lysis buffer? I have seen these referenced in various papers but would appreciate input whether you think it will help with processing planarian tissue (which is overall tougher than C elegans tissue, but relatively soft).
Thanks for your time!
Edited by bramwell, 18 July 2012 - 06:25 AM.